Antigen of hybrid M protein and carrier for group A streptococcal vaccine

ABSTRACT

Recombinant hybrid streptococcal M protein antigens are provided which elicit protective antibodies against Group A streptococci and prevent rheumatic fever. Recombinant hybrid genes which encode the antigen are provided. Vaccine compositions and methods of administering the compositions are provided to elicit immunity against Group A streptococci.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 08/914,479, filed Aug. 19, 1997, now allowed; which is acontinuation of U.S. patent application Ser. No. 08/409,270, filed Mar.23, 1995, now abandoned; which is a continuation of U.S. patentapplication Ser. No. 07/945,860, filed Sep. 16, 1992, now abandoned.These applications are incorporated herein by reference in theirentireties.

FIELD OF INVENTION

[0002] The present invention relates broadly to the field of recombinantvaccines. The vaccines are directed to preventing Group A streptococcalinfections, which may otherwise result in rheumatic fever.

BACKGROUND OF THE INVENTION

[0003] Acute rheumatic fever (ARF) is the major cause of heart diseasein children around the world. The disease is rampant in developingcountries where prevalence rates of rheumatic heart disease may be ashigh as 35-40 per thousand individuals. By one estimate, it affectsnearly six millon school-age children in India. Although the incidenceof ARF in the United States and other Western countries declinedmarkedly during the later half of the twentieth century, there has beena remarkable resurgence of the disease in the United States.

[0004] Streptococci are a group of bacteria with the capacity to grow inchains. Many varieties are part of the normal bacterial flora in humansand are not especially harmful. However, a particular subgroup ofstreptococcal bacteria, called Group A and represented by Streptococcuspyogenes, is a human pathogen. Between 20 and 30 millon cases of Group Astreptococcal infections occur every year in the United States alone.These cases include infections of the skin and throat, forms ofpneumonia and a recently identified disease resembling toxic shock. Themost common infection is acute streptococcal pharyngitis, or strepthroat, which occurs predominantly in school-age children. Strep throatqualifies as a major worldwide health problem if judged only by timelost from school and work and by the amount spent on related doctor'sfees.

[0005] Strep throat's toll is much greater, however. In as many as 4% ofthe pharyngitis cases that are untreated or treated ineffectively, thestrep infection leads to ARF. Current attempts to prevent ARF rely ontreatment of the pharyngitis with antibiotics. During a recent outbreakof ARF in Utah, only a fourth of the patients sought health care priorto the onset of symptoms, and only a third recalled a recent sorethroat. The finding that ARF may follow a subclinical infection in sucha high percentage of individuals and the fact that access to health carein developing countries is not widely available serve to underscore theneed for a safe and effective vaccine against Group A streptococci.

[0006] The causal relationship between streptococcal pharyngitis and ARFwas established over 50 years ago, yet the mechanism of the pathogenesisof the disease remains unclear. It is widely held that ARF is anautoimmune disease, and that in the susceptible host the infectiontriggers an immune response that leads to inflammatory and sometimesdestructive changes in target tissues. Streptococci have been shown tocontain antigens that are immunologically cross-reactive with hosttissues and heart cross-reactive antibodies from patients with rheumaticfever have been shown to react with streptococci. However, it was alsoshown that sera from patients with uncomplicated pharyngitis also maycontain heart cross-reactive antibodies, yet these patients do notdevelop clinical evidence of carditis. Until the significance of tissuecross-reactive antibodies in the pathogenesis of ARF is betterunderstood, there remains a need to exclude potentially harmful epitopesfrom vaccine preparations.

[0007] The surface M protein of Group A streptococci is the majorvirulence factor and protective antigen of these organisms. Group Astreptococci have developed a system for avoiding some of theantimicrobial defenses of a human host. Strains of streptococci that arerich in M protein evade phagocytosis by PMNs and multiply in non-immuneblood. Yet, resistance to an infection by these bacteria is possible ifthe host's body can produce opsonic antibodies directed against the Mprotein. Such antibodies will neutralize the protective capacity of theM protein and allow the streptococcus to be engulfed and destroyed byphagocytes. The development of secretory or mucosal immunity is also nowsuspected of playing an important role in preventing streptococcalinfections.

[0008] A major obstacle to effective vaccine development has been thetremendous number of M protein serotypes (now over 80). Laboratory testssuggest that antibodies against one serotype do not offer protectionagainst others. Immunity then appears to be type or sero-specific andoptimal vaccines would require that most of the serotypes berepresented, There is evidence that not all serotypes of Group Astreptococci have the same potential to trigger acute rheumatic fever insusceptible individuals. The concept of “rheumatogenic” and“non-rheumatogenic” organisms is supported by multiple surveillancestudies over many years and in diverse areas of the world. Thus, thereare probably about 12-15 serotypes responsible for most cases of ARF.Some of these are types 1, 3, 5, 6, 14, 18, 19,24,27 and 29.

[0009] Previous studies have shown that in many cases the protectiveepitopes of M protein may be separated from the potentially harmful,autoimmune epitopes of the molecule. The NH₂-terminal segments of Mproteins have evoked antibodies with the greatest bactericidal activity.

[0010] Previous studies have also shown chat synthetic peptides copyinglimited regions of types 5, 6 and 24 M proteins evoked type-specific,opsonic antibodies that were not heart tissue cross-reactive. Because oftheir lack of immunogenicity (haptens), the synthetic peptides werechemically linked covalently to carrier proteins. However, suchfragments of M proteins linked to carrier proteins with chemicalreagents do not result in hybrid proteins of defined structures. Thus,in general it has not been possible to obtain antigens which can elicitspecific, desired antibodies or which decrease the risk of undesirableside reactions. Further, formation of hapten—carrier complexes usingchemical cross-linking reagents is time-consuming and costly and resultsin undefined heterogenous mixtures of vaccine components.

[0011] It is evident from this description of the state of the art thatthere is an important need for a vaccine which is effective by raisingsero-specific antibodies against the various serotypes of Group Astreptococci, especially those serotypes capable of triggering acuterheumatic fever, which is known to follow a sore throat, withouteliciting cross-reaction with human tissue. Particularly, there is animportant need for a vaccine which has not only these properties, butwhich also is capable of raising protective antibodies to prevent sorethroat, skin infections, deep tissue infections and streptococcalinfections of the like that are not necessarily followed by rheumaticfever. The invention contributes to solving these important needs inhuman health.

SUMMARY OF THE INVENTION

[0012] The parent patent application is related to and was co-filed onthe same day as patent application Ser. No. 07/945,954, entitled“RECOMBINANT MULTIVALENT M PROTEIN VACCINE” with named inventors JamesB. Dale and James W. Lederer.

[0013] The present invention provides recombinant M protein antigens.The antigens are constructed by recombinant DNA methods. They arecomprised of amino acid fragments of serotypes of M protein, whichfragments carry one or more epitopes that evoke opsonic antibodiesagainst specific serotypes of Group A streptococcus and, if desired,when the fragments carry appropriate epitopes, also evoke protectiveantibodies. The fragments are either fused directly or linked in tandemby an amino acid linker to an appropriate carrier. The antigens aregenerally non-immunogenic (or not adequately immunogenic) because oftheir molecular size or for other reasons.

[0014] The invention thus provides a recombinant fusion antigencomprising a gene encoding the carrier protein and an NH₂ orCOOH-terminal M protein fragment carrying one or more epitopes. Therecombinant antigen does not elicit antibodies which cross react withhuman heart or other human tissue.

[0015] In accordance with the invention, there are provided mixtures ofantigens which are serotype-specific comprising the same or differentcarrier. Such a mixture of selected antigens-carriers or “cocktail”provides immunogenicity against several serotypes (and if desired raisedifferent protective antibodies). The recombinant fusion antigens areconstituted of segments of the NH₂ terminal portions of the M proteins,which fragments raise specific opsonic antibodies. Fusion antigens arealso provided which are constituted of the COOH-terminal fragments ofthe M proteins. The COOH-terminal fragments raise protective antibodiesof the mucosal or secretory type. In the antigen with an amino acidlinker, the carrier and the fragment of the M protein, which carries thedesired epitope, are linked in tandem by an amino acid linker, describedin greater detail hereinafter, which has the capacity to promote theconformation of the fragment of the M protein to optimize the exposureof the epitope and thus to optimally raise the desired antibodies.

[0016] The invention also provides for an antigen comprised of acarrier, which constitutes the carboxy-terminal portion of a serotype ofM protein linked by a linker or fused directly to an amino acid fragmentof M protein. The carrier and fragment may be of the same or differentserotype.

[0017] The invention also provides for carriers which are free ofepitopes which elicit antibodies to serotypes of streptococcal Mprotein.

[0018] The invention provides recombinant hybrid genes which nucleotidesequences encode for the antigens of the present invention and a methodof construction of such genes.

[0019] The invention further provides the new fusion genes or DNAfragments which code for the hybrid antigens and the transformedmicroorganisms (eukaryotes or prokaryotes) that express the hybridantigens.

[0020] The invention also provides avirulent microorganisms which aretransformed with the genes of the present invention. Thesemicroorganisms are especially suitable for oral administration to andimmunization of mammals, in particular humans.

[0021] The invention provides for methods of administration of theantigens of the present invention in therapeutic compositions via oral,intranasal and parenteral routes of administration, to induce or evokeopsonic and/or protective antibodies against serotypes of Group Astreptococcus. The administered compositions confer immunity toimmunized mammals against Group A streptococci.

[0022] The invention provides vaccine compositions which are comprisedof the antigens of the present invention and biologically acceptablediluents for administration to and immunization of mammals, inparticular humans. The composition is administrable orally, whereby theantigens are released from the transformed microorganism and the desiredantibodies are elicited, intranasally and parenterally.

[0023] The invention also provides for broad spectrum protection andwide-ranging immunity against all serotypes of Group A streptococci,particularly rheumatogenic streptococci by the formulation ofcompositions of the antigens, either singly or in mixtures or“cocktails”.

BRIEF DESCRIPTION OF THE FIGURES

[0024]FIG. 1 shows the DNA (SEQ ID NO:1) and deduced protein sequence(SEQ ID NO:2) of LT-B-M24 hybrid molecule.

[0025]FIG. 2 shows the immunoblot analysis of purified LT-B-M24 hybridprotein.

[0026]FIG. 3 shows the immunogenicity of LT-B-M24 in rabbits, asdetermined by ELISA.

[0027]FIGS. 4A and 4B show the order of the nucleotides (SEQ ID NO:3)and amino acid residues (SEQ ID NO:4) of an antigen of a fragment of M5and a carrier of the carboxy-terminal portion of M5.

[0028]FIGS. 5A and 5B show the order of the nucleotides (SEQ ID NO:5)and amino acid residues (SEQ ID NO:6) of an antigen of fragments of M5and a carrier of the carboxy-terminal portion of M5.

DETAILED DESCRIPTION OF THE FIGURES

[0029] The present invention and many of the attendant advantages of theinvention will be better understood upon a reading of the followingdetailed description when considered in connection with the accompanyingdrawings wherein:

[0030]FIG. 1 shows the DNA (SEQ ID NO:1) and deduced protein sequence(SEQ ID NO:2) of LT-B-M24 hybrid molecule. The sequence of the fusiongene was confirmed from base 228 to the 3′ end. The remainder of theLT-B sequence is from Clements (16). The NcoI site linking the LT-B andM24 components is indicated. The M24 subunit is underlined.

[0031]FIG. 2 shows the immunoblot analysis of purified LT-B-M24 hybridprotein. The purified protein was electrophoresed on anSDS-polyacrylamide gel and transferred to nitrocellulose paper.Coomassie blue stained a single band with an apparent molecular weightof 14 kDa (lane A). The purified protein reacted with rabbit antiseraagainst LT-B (lane B) and SM24 (1-29)C (lane C).

[0032]FIG. 3 shows immunogenicity of LT-B-M24 in rabbits, as determinedby ELISA. Three rabbits (∘, , Δ) were immunized with 300 μg LT-B-M24 attime 0 and at 4 weeks (arrows) and sera collected at two-week intervalswere assayed for the presence of antibodies against pep M24 by ELISA.Titers are expressed as the reciprocal of last dilution of antiserumthat resulted in an O.D. of >0.1 at 580 nm. ELISA performed at variousintervals after the initial injection of LT-B-M24 revealed a briskantibody response in all three rabbits, even after a singleintracutaneous dose of LT-B-M24.

[0033]FIGS. 4A and 4B show a construct of 762 nucleotides (SEQ ID NO:3)coding for an antigen of 254 amino acid residues (SEQ ID NO:4), asshown. The antigen is comprised of an M5 hapten fragment of 16 aminoacids. The fragment is joined by a BamH1 restriction site to a carrier,which is the carboxy-terminal half of M5. The carrier includes 2.5C-repeats, which each commence with the tetrapeptide Asn-Lys-Ile-Ser(SEQ ID NO:19). While there is no amino acid linker linking the fragmentand carrier, the BamH1 is a suitable site for insertion of anappropriate linker.

[0034]FIGS. 5A and 5B show a construct of 852 nucleotides (SEQ ID NO:5)coding for an antigen of 284 amino acid residues (SEQ ID NO:6), asshown. The antigen is comprised of three segments of M5 designated A, Band C, respectively. The C segment is joined by a BamH1 restriction siteto a carrier, which is the carboxy-terminal half of M5. The carrierincludes 2.5 C-repeats, which each commence with the tetrapeptideAsn-Lys-Ile-Ser (SEQ ID NO:19). While there is no amino acid linkerlinking the fragment and carrier, the BamH1 is a suitable site forinsertion of an appropriate linker.

DETAILED DESCRIPTION OF THE INVENTION

[0035] The above and various other aspects and advantages of the presentinvention are described hereinafter. All references above andhereinafter are incorporated by reference, and identified in theBibliography as numbered in the text.

[0036] The invention provides for a recombinant hybrid streptococcal Mprotein antigen which elicits opsonic antibodies to a serotype of GroupA streptococcus, which comprises a carrier linked by an amino acidlinker to an amino acid fragment, the fragment having at least oneepitope of a serotype of Group A streptococcal M protein. The fragmentelicits opsonic antibodies to the epitope or epitopes of the targetserotype, without eliciting cross-reactive antibodies to mammalian hearttissue antigens.

[0037] It is also an embodiment of the present invention that theantigen of carrier and fragment are joined or fused together without thepresence of a linker.

[0038] The amino acid fragment is of a portion of a streptococcal Mprotein serotype of Group A streptococcus. The fragment is of a peptidesize which is non-immunogenic (or inadequately immunogenic) by itself.In that sense, the fragment may be considered a hapten if it wereconsidered by itself. Where a hapten is defined as a low molecularweight determinant group, which by itself is non-immunogenic, but whichbecomes so when placed on a larger “carrier” molecule. If coupled to acarrier protein of appropriate size, haptens are capable of inducing astrong immune response in an animal or human.

[0039] By the present invention, the recombinant hybrid proteins areselected to be constituted of amino-terminal (NH₂) fragments of Mproteins which contain immunoprotective epitopes, which elicit opsonicantibodies to a target serotype of Group A streptococcal M protein. Theamino terminal fragment of M protein is constituted of the first 35amino terminal residues of the M protein. These fragments of the presentinvention do not contain cross-reactive or autoimmune epitopes togenerate an autoimmune response directed to heart or other tissueantigens. As a consequence, the risk of developing ARF is minimized. Thefragments of the M protein elicit antibodies of the opsonic or serumtype which assist the phagocytic engulfment of the Group Astreptococcus. The fragments of the M proteins may also elicit, if sodesired, protective antibodies, which are secretory or mucosalantibodies, (e.g. from the saliva or nasal membranes), which play acritical role in a first-line defense against an invading streptococcusat the locus or site of infection. In that instance, the fragments areportions of the COOH-terminal half of M proteins, which fragments arefree of epitopes which elicit antibodies which cross-react with heart orother tissues.

[0040] Thus two types of antibodies are elicited from epitopes locatedon two different portions of the M protein molecule. The NH₂-terminalfragments contain epitopes which elicit serum or opsonic antibodies inimmunized mammals. The COOH-terminal fragments of the M protein elicitprotective secretory or mucosal immunity in immunized mammals, e.g. IgAantibodies.

[0041] It should be noted in conjunction with the invention, as has beendescribed herein, that not all M protein epitopes are sero-specific intheir NH₂-terminal portion. Some epitopes of particular serotypes, suchas M5, do cross-react to some extent with streptococci of a type otherthan M5, such as M6 or M19. And to some extent, this also occurs withother M serotypes. Accordingly, it is within the scope of the inventionthat when type-specificity is referred to, this does not exclude somecross-reactivity between certain shared structures of differentserotypes. However, one should be careful to note that such sharedepitopes that would cross-react with heart tissue are to be excludedfrom the selected fragments. Also included are NH-terminal fragmentswhich, in addition to evoking opsonic antibodies, also contain epitopesthat evoke protective secretory or mucosal antibodies.

[0042] A mixture or cocktail of antigens is provided. The antigens inthe mixture contain NH₂ or COOH-terminal fragments of M protein ofdifferent serotypes which can elicit opsonic and protective antibodies,respectively, to a wide range of serotypes, particularly therheumatogenic streptococci.

[0043] Thus, if it is desired in accordance with the invention toprovide immunity against streptococcus infections that are likely tocause rheumatic fever, DNA molecules will be constructed which code forNH₂-or COOH-terminal fragments which contain epitopes that elicitopsonic or protective to antibodies types 1, 3, 5, 6, 14, 18, 19, 24, 27or 29 M proteins, for instance and linked in tandem by an amino acidlinker or fused directly with an appropriate carrier.

[0044] Suitable portions of NH₂-terminal fragments are from 10 to 35amino acid residues in length. Fragments of this length have been shownto elicit opsonic antibodies but not to contain heart-cross reactiveepitopes. Any portion of the amino terminal fragment is suitable as longas the fragment, when constituting a portion of the antigen of theinvention, will elicit opsonic antibodies. An example of a suitableamino terminal portion is that portion containing amino acid residues 14to 26 of the amino terminal fragment of the M protein.

[0045] Examples of suitable NH₂-terminal fragments of M protein forconstructing antigens, which elicit opsonic antibodies in an immunizedanimal are described hereinafter. For M24, there is provided a 15 aminoacid fragment of the orderVal-Ala-Thr-Arg-Ser-Gln-Thr-Asp-Thr-Leu-Glu-Lys-Val-Gln-Glu (SEQ IDNO:7). For M5, there is provided a 15 amino acid fragment of the orderAla-Val-Thr-Arg-Gly-Thr-Ile-Asn-Asp-Pro-Gln-Arg-Ala-Lys-Glu (SEQ IDNO:8). For M6, there is provided a 15 amino acid fragment of the orderArg-Val-Phe-Pro-Arg-Gly-Thr-Val-Glu-Asn-Pro-Asp-Lys-Ala-Arg (SEQ IDNO:9). For M19, there is provided a 15 amino acid fragment of the orderArg-Val-Arg-Tyr-Thr-Arg-His-Thr-Pro-Glu-Asp-Lys-Leu-Lys-Lys (SEQ IDNO:10). For M3, there is provided a 15 amino acid fragment of the orderAsp-Ala-Arg-Ser-Val-Asn-Gly-Glu-Phe-Pro-Arg-His-Val-Lys-Leu (SEQ IDNO:11). For M1, there is provided a 15 amino acid fragment of the orderAsn-Gly-Asp-Gly-Asn-Pro-Arg-Glu-Val-Ile-Glu-Asp-Leu-Ala-Ala (SEQ IDNO:12). For M18, there is provided a 15 amino acid fragment in the orderAla-Pro-Leu-Thr-Arg-Ala-Thr-Ala-Asp-Asn-Lys-Asp-Glu-Leu-Ile (SEQ IDNO:13). For M12, there is provided a 15 amino acid fragment of the orderHis-Ser-Asp-Leu-Val-Ala-Glu-Lys-Glu-Arg-Leu-Glu-Asp-Leu-Gly (SEQ IDNO:14). These NH₂-terminal fragments will elicit opsonic antibodies inimmunized animals when linked or fused to an appropriate carrier, whichcarrier may also favorably elicit desirable antibodies. These, as wellas other antigens of the present invention may be administered to amammal as a cocktail or mixture to elicit broad-spectrum antibodies. Insuch fashion, broad spectrum immunity is provided to immunized mammalsagainst Group A streptococci.

[0046] It is important to note that the invention is not limited to aparticular amino acid sequence, wherever amino acid sequences arereferred to or described, as above. In any particular amino acidsequence or fragment referred to herein, any one or more amino acids canbe removed, substituted or replaced by some other amino acid(s),providing that the desired epitopes are not adversely affected by suchchanges in the primary structure of the protein fragments. Indeed, thisis a quite common occurrence for the M protein among various strainswithin the same serotype. This tendency of variation in the sequence ofthe M protein has been shown for several such M proteins.

[0047] Accordingly, any single fragment of a given serotype encoded by agene may have its sequence altered, so as to carry multiple epitopes fora multitude of strains within the same serotype. In this fashion, asingle antigen will elicit desirable antibodies to a number of strainswithin the same serotype. It is therefore an important concept of theinvention that once reference is made to a particular serotype (i.e., ofany one of the known or to be discovered serotypes, e.g., 1-82),reference is not intended to one single strain of a type, but to thevarious strains within the serotype. Thus, not only is it a fundamentalconcept of the invention to provide a vaccine of a cocktail or mixtureagainst different serotypes, but also a vaccine directed to differentstrains within that particular serotype.

[0048] Thus, in accordance with the invention, there is provided avaccine which is effective against not only different serotypes of Mproteins, but also against various strains within the individualserotypes.

[0049] The amino acid linker sequences ideally contribute to the maximumaccessibility of the epitopes of the fragments so as to generate thedesired antibodies. Thus the linkers may function to influence theorientation, conformation or other aspects of the amino acid fragment-carrier molecule. Ideally, the linkers contribute to the orientation sothat the hybrid molecule, or at least the amino acid fragment, mimicsthe native molecule.

[0050] The linker amino acid sequence is provided by construction of agene coding for the desired M protein amino acid sequence and carrierwith a restriction site to permit insertion of the DNA coding for thelinker between the sequences of the fragment and the carrier, whichlinker links the carrier and fragment in tandem. Illustratively, such alinker is a proline-rich linker of Pro-Gly-Asn-Pro-Ala-Val-Pro (SEQ IDNO:15). Other amino acid linkers may be used such as Ile-Pro-Gly,Asp-Pro-Arg-Val-Pro-Ser-Ser (SEQ ID NO:16) or His-Gly. An amino acidlinker of Asp-Pro-Arg-Val-Pro-Ser-Ser (SEQ ID NO:16) or its equivalentis also considered suitable (SEQ ID NO:18). While in theory, a linkercould be constituted by one amino acid, so long as the desiredimmunoreactive conformation is achieved, longer linker regions may bemore suitable to optimize the immunogenicity of the epitopes.

[0051] The effect of the different linkers on the immunogenicity of thehybrid molecule may justify further investigations. It is not excludedthat, depending on the type and number of the amino acids of the linker,a hybrid antigen of ideal or close to ideal high immunogenicity may beidentified.

[0052] A linker of hydrophobic acids is most desirable, such astryptophan, alanine, leucine, isoleucine, valine, tyrosine,phenylalanine, proline, methionine and combinations thereof. The linkersmay range in number from 2 to 30, providing that there is no adverseeffect on the immunogenicity of the molecule. Quite satisfactory resultshave been obtained with a two amino acid linker of His-Gly, suggestingthat such a small molecule may be satisfactory for M protein components.The sequence of the amino acids in any particular linker does not appearat this time to be critical.

[0053] As described further below, linkers are useful but not essentialto join the carrier and fragment. The carrier and fragment may be joinedor fused together directly.

[0054] According to the present invention, gene constructs are providedwhich encode an amino-terminal fragment of an M protein serotype linkedwith the carboxy-terminal half of an M protein, which functions as acarrier. See FIGS. 4A-B and 5A-B (SEQ ID NOS: 3,4,5, and 6,respectively). As shown therein, a BamH1 restriction site serves as ajunction between the fragment and carrier. It is contemplated and withinthe scope of the invention that the restriction site be a suitable sitefor insertion of an appropriate amino acid linker. The carboxy-terminalcarrier may elicit protective mucosal or secretory antibodies toserotypes of M protein. These antibodies may play an important role inpreventing colonization and infection by Group A streptococci. Such acarrier thus serves a dual purpose. It serves the function ofintroducing the epitope(s) present on the amino-terminal hapten fragmentto the macrophages for processing and antigen presentation to helper Tcells for generating a type-specific humoral response. And the carrierpossesses epitopes which may elicit more broadly protective IgAantibodies at the mucosal or secretory level. These antibodies are morebroadly protective than the type-specific opsonic type, as they sharemore homologous epitopes among the various types of rheumatogenicstreptococci. In this fashion, such protective antibodies areadvantageously of a cross-protective or cross-serotype nature.

[0055] Instead of using the entire carboxyl-terminal of any one of theserotypes, it may be advantageous and desirable to only use one or moreC-repeats of the M protein as a carrier. It is noteworthy that thecarboxyl-terminal portion of M protein used in the construct need not beone of the same serotype as that which constitutes the amino terminalportion of the construct.

[0056] Instead of using the COOH-terminal portion or half of an Mprotein as a carrier, other suitable carriers for the fragments includesurface proteins from gram-positive cocci (mostly from streptococci andstaphylococci) (32). These proteins, which have been sequenced, are IgAbinding protein (ARP 4), F_(c) binding protein (F_(c)RA), human IgGF_(c) binding protein (Protein H), C5a peptidase (SCP) and T6 surfaceprotein from S. pyogenes; wall-associated protein (wap A) and cellsurface protein (PAc and spa P) from S. mutans: Protein G, an IgGbinding protein from Group G streptococci, Protein A and fibronectinbinding protein (FaBP) from S. aureus, and a cell wall protease (wg 2)from S. cremoris. Beginning at the C-terminal end of these molecules,these proteins and the M proteins all have a similar arrangement ofamino acids. Up to seven charged amino acids are found at the C-terminuswhich are composed of a mixture of both negative and positive chargedresidues. Immediately, N-terminal to this short charged region is asegment of 15-22 predominately hydrophobic amino acids. Beginning aboutnine amino acids N-terminal from the hydrophobic domain is found ahexapeptide with the consensus sequence LPSTGE (SEQ ID NO: 17), that isextremely conserved among all the proteins. Analysis of 12 of thesesurface molecules revealed that these proteins contain repeat segments,as in the M proteins, and were predominately helical within the regioncontaining the repeat segments.

[0057] These proteins, as carriers, can be linked in tandem by an aminoacid linker to NH₂ or COOH-terminal fragments or fused directly with thefragments, so long as the immunogenicity of the antigens is notadversely affected.

[0058] The amino acid fragments of the present invention are linked,either with or without a linker, to an appropriate carrier. For thispurpose, any type of carrier is contemplated so long as the amino acidfragment linked to the carrier generates an opsonic or protective immuneresponse to the epitopes of the fragment. The carrier may be a moleculewhich is free of an epitope which elicits antibodies to a serotype ofstreptococcal M protein. An example would be the B subunit E. colilabile toxin (LT-B). Alternatively, the carrier may be selected from theCOOH-terminal portion of the M protein which is not cross-reactive withhuman tissue, and which may favorably elicit protective mucosalantibodies. Or the carrier may be the COOH-terminal portion of surfaceproteins of gram-positive cocci, as described above.

[0059] Other examples of suitable carriers may be keyhole limpethemocyanin (KLH), tetanus toxoid, diphtheria toxoid, bovine serumalbumin, hen egg lysozyme, gelatin, bovine gammaglobulin and flagellinpolymer.

[0060] As shown in FIGS. 4A-B (SEQ ID NOS: 3 and 4, respectively) and5A-B (SEQ ID NOS: 5 and 6, respectively), it is an embodiment of thepresent invention that the presence of one or more linker in the geneconstructs, linking in tandem the fragment and carrier, is not requiredto elicit opsonic or protective antibodies in an immunized host. Thefragment and carrier, in this embodiment of the invention, are fuseddirectly to each other. A fusion antigen is suitable so long as theimmunogenicity of the antigen is not adversely affected. The resultinggene constructs are fusion constructs.

[0061] Alternatively, a linker of amino acids may be added by chemicalmeans after the antigen is expressed, e.g., by treatment withsuccinimidyl-4-(N-maleiamido-methyl) cyclohexone-1-carboxylate.

[0062] It is also contemplated that the constructs of the invention beconstructed to contain a fragment of the NH₂ or COOH-terminal region ofserotypes, which are not known to have a rheumatogenic effect, as thosetypes described above and in the literature. In those instances, wheresuch fragments have not yet been sequenced or not yet been published,whether rheumatogenic or non-rheumatogenic, one skilled in the art cansequence such structures by methods readily available. The inventionalso includes the construction of hybrid constructs containing repeatingamino-terminal or carboxyl-terminal M protein fragments using PCR. Oneskilled is the art can utilize homologous regions of published M proteinemm gene sequences from Group A streptococci (GAS; Streptococcuspyogenes) to design three primer pairs for PCR and three oligonucleotideprobe sequences internal to the amplified products. One set of primersand corresponding probe can detect and lead to amplification of emm(-like) genes of virtually every type (“all M”). Another set (“SOR⁻M”)amplifies only emm (-like) genes from GAS negative for serum opacityreaction (SOR). And a third set (“SOR⁻M”) expands only emm (-like) genesfrom SOR-GAS. Using the “all M” primer pair for PCR on the genomic DNAfrom gas of 29 different M types, as well as from a Group C and a GroupG streptococcal isolate, DNA fragments within the expected size rangewere amplified in every assay. All PCR products reacted with the “all M”probe.

[0063] Thus, the invention contemplates a hybrid fragment-carrierprotein antigen encoded by an appropriate gene or genes to express in anappropriate organism, the antigen that will elicit the desiredantibodies. Thus encompassed within the scope of the present inventionare antigens comprised of opsonic antibody-generating NH₂ orCOOH-terminal fragments of M protein from all the known rheumatogenictypes of streptococci, and fragments from types of streptococci whichare not, or at least not yet known or shown to be, associated with ARF.An example of a non-rheumatogenic streptococcal type, the M proteinantigen of which is within the scope of the invention, is type 12.

[0064] The appropriate genes of the present invention are constructedand expressed, as described hereinafter. The genes encoding theappropriate carriers of the present invention are inserted intoappropriate plasmids. Non-limiting examples of the appropriate carriersare the carboxyl-terminal half of M proteins, the carboxyl-terminal halfof surface proteins of gram-positive cocci and the B subunits of E. colilabile (LT-B) and cholera toxin (CT-B) from Vibrio cholerae. Theplasmids are modified to contain a small polylinker with threeendonuclease restriction sites at the 3′ end, followed by transcriptionterminators in each reading frame. The selected genes encoding thedesired NH₂ or COOH-terminal fragments of M protein are constructed in asuitable manner. For instance, a pair of oligonucleotides coding for thefragments are synthesized using an automated DNA synthesizer (ABI, mode1381A). The desired oligonucleotides copy the appropriate first numberof base pairs of the genes encoding the desired NH₂ or COOH-terminalfragments of M protein. Additionally, the oligonucleotides encompass theright hand side of an appropriate restriction site at the 5′ end, forinstance NcoI. The oligonucleotides are mixed in equimolar ratios,heated to an appropriate temperature and allowed to anneal at ambienttemperature. The appropriate plasmids are digested with restrictionenzymes, and the cut plasmids are then purified. For instance, plasmidpPx1604 was digested with NcoI and EcoRV and purified from agarose gelsover glassmilk (Geneclean, Bio 101, La Jolla, Calif.). The syntheticoligonucleotide pairs of interest are then ligated into the cut sites ofthe plasmids. The plasmids containing the M protein fragments ofinterest are then used to transform an appropriate microorganism. Forinstance, E. coli JM105 is a suitable microorganism. Transformants arethen screened by an appropriate method, e.g., dot blot analysis usingappropriate antisera.

[0065] For high level expression of the M protein antigens of thepresent invention, insertion of the selected gene constructs, encodingthe antigens, into suitable plasmids is carried out. An example of asuitable plasmid is pK K223-3 (Pharmacia, Uppsala, Sweden). The genesare cut from suitable plasmids, for instance pPX1604, with appropriaterestriction enzymes. Suitable enzymes are EcoRI and SalI. The selectedgenes are purified by cutting from agarose gels. Klenow fragment is usedto end repair the purified DNA. The purified gene constructs are cutwith suitable restriction enzymes, for instance EcoRI. The cut geneconstructs are then ligated into the appropriate restriction sites ofselected high expression plasmids. For instance, the cut genes areligated into the EcoRI and SmaI restriction enzyme sites of pKK223-3plasmids. The selected plasmids carrying the gene constructs of thepresent invention are then used to transform suitable microorganisms.For example, E. coli JM105 is transformed with the selected plasmids.Expression of the proteins is detected in a suitable fashion, such as bydot blot analysis using appropriate antisera. For example, the desiredtransformants were screened for expression of the gene encoding aNH₂-terminal fragment’ of M24-LT-B carrier (subunit B of E. coli labiletoxin) by dot blot analysis using rabbit antisera against a syntheticpeptide of M24, SM24 (1-29) C and rabbit antiserum against purified LT-B(16), kindly provided by Dr. John Clements of Tulane University. Theappropriate positive transformants harboring the selected plasmidscarrying the genes of the present invention are selected for expressionand purification of the recombinant protein antigens of the presentinvention.

[0066] The vaccine compositions of the invention include the antigens ofthe invention and biologically acceptable diluents or adjuvants. Thecompositions are suitable for eliciting opsonic and/or protectiveantibodies to serotypes of M protein of Group A streptococcus. Theadministered compositions of the present invention elicit antibodies,without eliciting cross-reactive antibodies to mammalian heart tissueantigens.

[0067] Appropriate biologically acceptable diluents or adjuvants for thepresent composition may be selected from a wide group of such diluentsor adjuvants as readily known to one of skill in the art. A non-limitingexample of a diluent is phosphate-buffered saline. The compositions maybe administered singly or as a mixture or cocktail.

[0068] Another aspect of the present invention are hybrid or fusiongenes which have been constructed which encode the antigens of thepresent invention. The fusion genes code for the antigens of theinvention, constituted as described above, of amino acid fragmentslinked to the selected carrier. The genes are inserted into suitableself-replicating vehicles, like plasmids. The plasmids containing thegenes are then used to transform nonvirulent microorganisms. Thetransformed microorganisms express the hybrid or fusion protein antigenswhich are capable of eliciting opsonic and/or protective antibodiesagainst serotypes of Group A streptococcus in immunized mammals, withouteliciting cross-reactive antibodies to mammalian heart tissue antigens.

[0069] One method provides for administration of the compositions tomammals, in particular humans, to elicit opsonic and/or protectiveantibodies directed to epitopes present in the hybrid antigens of thepresent invention. No antibodies cross-reactive with heart tissueantigens are elicited. The method comprises administering orally to saidmammal, in an amount effective to confer immunity against Group Astreptococci infection, a therapeutic composition which comprises abiologically acceptable carrier and a non-virulent, live bacterium asdescribed in U.S. Pat. No. 5,124,153 to Beachey et al., and allreferences therein incorporated herein by reference. The bacterium istransformed with a plasmid encoding and expressing an antigen of thepresent invention. The antigen is released from the bacterium, wherebyprotective antibodies to the antigen of the same serotype as theimmunizing antigen are elicited, without eliciting antibodies which arecross-reactive with heart tissue antigens. Immunity against streptococciinfection is thereby conferred to the mammal.

[0070] The present invention encompasses administering orally multipletherapeutic compositions. Each composition comprises a hybrid antigen ofa serotype of streptococcis. The compositions may be administeredindividually or as a mixture or cocktail of several compositions. Inthis fashion, a mammal is immunized against one or more rheumatogenicserotypes of Group A streptococcus. Broad spectrum protective immunitymay therefore be established against all rheumatogenic streptococci.

[0071] Any biologically acceptable carrier may be used. A biologicallyacceptable carrier may be PBS, as a non-limiting example. Particularlypreferred is a dose of the therapeutic composition suspended in 25 ml ofPBS, pH 7.2 containing 5 mg/ml kanamycin sulfate and 1 mg/ml each ofparaaminobenzoic acid (PABA) and 2,3-dihydrobenzoic acid (DHB).

[0072] In accordance with the invention, it is preferable that theplasmids which encode the M protein genes of the present invention becloned first and expressed in Escherichia coli. Any other entericbacilli of the coliform group such as Klebsiella or Enterobacter can beused, but normally E. coli is preferred. Therefore the plasmid carryingthe M gene is isolated and purified and then a construct is built totransform the desired non-virulent bacteria, such as the araA-S.typhimurium (SL3261). It is to be noted that this mutant strain exhibitsa nutritional marker both for PABA and 2,3-DHB. It is to be noted thatanother desired specie of S. typhimurium is recA- S. typhimurium,particularly strain Ty21a (17).

[0073] It may be desired to obtain the M protein gene from a virulentstrain of S. pyogenes. However, it is preferable to obtain the gene froman attenuated, non-virulent strain of S. pyogenes, or to fabricate thenucleotide sequence coding for the desired M protein.

[0074] The recombinant DNA cloning vectors of the present invention arenot limited for use in a single species or strain of Salmonella. To thecontrary, the vectors are broadly applicable and can be transformed inhost cells of other gram negative bacteria such as of theEnterobacteriaceae genus (such as Shigella and Klebsiella like(Klebsiella pneumoniae; Enterobacter like Enterobacter aerogenes.Salmonellae, such as Salmonella arizona, and Citrobacter may be used ifappropriately rendered non-virulent or attenuated.

[0075] Common Salmonella species which may be used when attenuated andrendered non-virulent include the following: S. paratyphi A, S.schottmulleri, S. typhimurium, S. paratyphi C, S. choleraesuis, S.Montevideo, S. newport, S. typhi, S. enteritidis, S. gallinarum, and S.anatum.

[0076] In accordance with the invention there may also be used as hostfor the recombinant DNA cloning vectors of the present inventionbacteria of the Streptoccus genus which are non-virulent or which havebeen made non-virulent or attenuated, including streptococci of theimmunological groups A-O but generally other than A. SuitableStreptococci which can be used as bacterial host include S. cremoris, S.faecalis, S. salivarius, S. mitior, S. mitis, S. mutans and S. sanguis.Particularly preferred is S. mutans which is non-cariogenic.

[0077] Additional appropriate microorganisms which may be attenuated andtransformed in accordance with the invention are known.

[0078] Generally any enteric bacterium may serve as the host bacterium.It is preferable that the host bacterium only survive in the subjectlong enough to elicit the opsonic response, but generally any bacterialstrain that has been attenuated so as not to colonize yet still multiplyto a limited degree to elicit antibodies to the protein antigen of thepresent invention can be used. In a preferred embodiment of theinvention the Aro⁻ strain of S. typhimurium is used, which requires twometabolites not found in mammalian tissues, PABA and 2,3-DHB. As aresult, the inoculated bacteria die after several generations from alack of these metabolites.

[0079] However, any mutated microbial agent with a metabolic deficiencyfor nutritional compounds not found in the tissues of the subject to beimmunized, or one so made by genetic manipulations, may be employed.

[0080] It is to be noted that the non-virulent aro⁻ Salmonellatyphimurium SL3261 which has been transformed with a plasmid containinga recombinant hybrid gene encoding a protein antigen expressed the M5protein molecule, which expression is confined almost exclusively to theS. typhimurium cytoplasmic compartment. It is unique and unexpectedaspect of this invention that an immunogenic and protective surfaceantigen such as the Streptococcal M protein antigen is expressed in thecytoplasm of the non-virulent host bacterium.

[0081] Thus it can be seen that in accordance with the invention, thedesired nucleotide sequence which codes for and expresses the proteinantigen, which is effective to elicit opsonic and/or protectiveantibodies to streptococcal serotypes, can be cloned into a variety ofhosts. In a broader sense therefore, the transformed host in which thenucleotide sequence is found after replication need not be heterologouswith respect to the nucleotide sequence, nor does the sequence need tobe heterologous with respect to the microorganisms.

[0082] In accordance with a specific embodiment of the method ofimmunization of a warm-blooded animal, it has been shown that a) peroraladministration of up to 1.65×10⁹ mutant non-virulent Salmonellacontaining the plasmid pMK207 encoding an antigen of serotype M5 waswell tolerated in mice; b) plasmid mPK207 was extremely stable both invitro and in vivo; c) the mice receiving the highest dose (10⁹) ofbacteria harbored the microorganisms in the liver for as long as threeweeks without ill effects; d) the mice immunized orally withnon-virulent transformed Salmonella expressing the gene developedopsonic serum antibodies as early as three weeks against serotype M5Streptococci; and e) the immunized mice were completely protected atthree weeks against intro-peritoneal challenges of the homologousserotype M5 (but not the heterologous serotype M24) Streptococci.

[0083] It is noteworthy that no cross-reactive immunity is observed whenthe composition of the invention is administered orally. The cytoplasmicexpression of the M protein antigen in the non-virulent bacterium isespecially advantageous for this oral administration. The antigen isprotected within the cytoplasm of the non-virulent bacterium from theacids of the stomach and other damaging agents until the non-virulentcell dies and releases the antigen, ordinarily in the small intestine,which is the preferred location for delivery of the antigens.

[0084] In accordance with the invention the non-virulent bacterium mayalso be used as a host for recombinant DNA cloning vectors containingnucleotide sequences which code for and express the immunogenic proteinantigens of the present invention which are specifically effective toconfer immunity against Streptococcal infections and which are notcross-reactive with human tissue antigens, especially those of theheart.

[0085] The therapeutic compositions of the present invention may also beadministered parenterally. Mammals, in particular humans, immunizedparenterally with a sufficient amount of the therapeutic composition ofthe present invention develop opsonic and/or protective antibodiesdirected to the epitopes of the hybrid streptococcal M protein antigen.Non-limiting examples of such parenteral routes of administration areintracutaneous and intramuscular.

[0086] For intracutaneous injection, 100-300 μg of hybrid antigenemulsified in complete or incomplete Freund's adjuvant was administeredin a mammal. A booster injection of about the same dose in saline wasadministered about one month later. Blood was obtained prior to thefirst injection and at two-week intervals thereafter for eight weeks.

[0087] A topical method of administration is also provided, namelyintranasal.

[0088] For intranasal administration, a mammal received about 50 μg toabout 10 mg of purified antigen in an appropriate diluent foradministration.

[0089] In accordance with the invention, the therapeutic composition maybe administered singly in series or advantageously in a mixture orcocktail of multiple compositions to elicit broad spectrum immunityversus Group A streptococci.

[0090] Other advantages of the invention will appear from thenon-limiting materials, methods and examples which follow.

EXAMPLE I

[0091] Purification of LT-B-M24 Hybrid Protein.

[0092] The recombinant LT-B-M24 protein was purified from cell extractsof JM105 (harboring pEC.LT-B-M24) grown overnight in one liter ofL-broth supplemented with 75 μg/ml ampicillin, 25 μg/ml streptomycin and1 mM isopropylthiogalactoside (IPTG, Bethesda Research Laboratories,Inc., Bethesda, Md.). -The cells were pelleted at 7,000×g andresuspended in 50 ml 100 mM carbonate buffer, pH 11, containing 100μg/ml lysozyme, 1 mM ethylenediaminetetraacetic acid (EDTA, SigmaChemical Co., St. Louis, Mo.) and 100 μg/ml phenylmethylsulfonylfluoride(PMSF, Sigma Chemical Co.) and incubated at 37° C. for 30 minutes. Thecells were centrifuged at 7,000×g and the supernatant was dialyzedagainst distilled water and lyophilized. Purification was performed byloading 50 mg of hybrid protein extract onto a preparativepolyacrylamide gel electrophoresis unit (Prep Cell, Model 491, Bio Rad.,Inc.) using a 37 mm column and a 9 cm 11% polyacrylamide gel. Six mlfractions were collected and assayed for the presence of recombinantprotein by Western blot analysis using rabbit antiserum against pep M24.Fractions containing activity were pooled, dialyzed and lyophilized.

EXAMPLE 2

[0093] Immunization of Rabbits.

[0094] Rabbits were immunized intracutaneously with 300 μg LT-B-M24protein emulsified in complete Freund's adjuvant. A booster injection ofthe same dose in saline was given four weeks later. Blood was obtainedprior to the first injection and at two-week intervals thereafter foreight weeks.

EXAMPLE 3

[0095] Assay for M Protein Antibodies.

[0096] Rabbit antisera were assayed for the presence of M proteinantibodies by ELISA using LT-B, pep M24 or SM24 (1-29)C as solid phaseantigens, as previously described. Opsonic antibodies against type 24streptococci were assayed by in vitro opsonophagocytosis tests. Briefly,0.1 ml of test serum was added to 50 μl of a standard suspension ofstreptococci. 0.4 ml heparinized, non-immune normal human blood wasadded and the mixture was rotated end-over-end for 30 minutes. Thepresence of opsonic antibodies was estimated by counting the percentageof neutrophils with associated streptococci (percent opsonization) onstained smears. Indirect bactericidal assays were performed using thesame mixture as described above except that fewer streptococci wereadded. The tubes were rotated for three hours and pour plates were madeusing 0.1 ml of the test mixture in 5% sheep blood agar. CFU ofstreptococci surviving were counted after incubating overnight at 37° C.

EXAMPLE 4

[0097] Assay for Heart- Crossreactive Antibodies.

[0098] Rabbit antisera against LT-B-M24 were screened for the presenceof heart-crossreactive antibodies by indirect immunofluorescence assaysusing thin sections (4 μ) of human myocardium, as previously described.

EXAMPLE 5

[0099] Mouse Protection Tests.

[0100] Passive mouse protection tests were performed as previouslydescribed. Briefly, Balb/c mice were injected intraperitoneally with 0.5ml test serum, and 24 hrs later, groups of mice were challengedintraperitoneally with 10-fold dilutions of type 24 streptococci. Pourplates were performed to determine the CFU of streptococci that eachgroup received. The LD₅₀ was calculated using the method of Reed andMuench.

EXAMPLE 6

[0101] Assay for M Protein Epitopes that Evoke Mucosal AntibodiesBroadly Protective Against Infection.

[0102] Rabbit antisera were screened for the presence of broadlyprotective antibodies using passive mouse protection assays. Antiserawere first tested for their ability to react with the surface M proteinof multiple heterologous serotypes of Group A streptococci by ELISA.Those that recognized M protein epitopes in their native conformationswere then used to passively protect mice against intranasal challengeinfections. Antibodies were absorbed to virulent streptococci and mirewere challenged intranasally with 10⁷ CFU. Throat cultures were obtainedon alternate days and deaths were counted over the ensuing 14 days.

[0103] By way of the Examples, it is shown that the antigens of thepresent invention elicit opsonic and/or protective antibodies directedto epitopes on the antigens, and confer immunity to immunized mammalsagainst Group A streptococci. The antigens may be advantageously mixedto form a cocktail. MATERIALS AND METHODS

[0104] Construction and expression of LT-B-M24 fusion gene. PlasmidpPX1604, which contains the gene for LT-B, was kindly provided by RobertBrey, Praxis Biologics, Rochester, N.Y. pPX1604 is a derivative ofpJC217 and was modified to contain a small polylinker with threeendonuclease restriction sites at the 3′ end followed by transcriptionterminators in each reading frame. The M24 component of the hybrid geneconsisted of a pair of oligonucleotides that were synthesized using anautomated DNA synthesizer (ABI, model 381A). The oligonucleotides copiedthe first 36 base pairs of the emm24 gene and included the right handside of an NcoI site on the 5′ end. The oligonucleotides were mixed inequimolar ratios in ligation buffer, heated to 65° and allowed to annealat ambient temperature. Plasmid pPX1604 was digested with NcoI and EcoRVand the cut plasmid was purified from agarose gels over glassmilk(Geneclean, Bio101, La Jolla, Calif.). The synthetic M24 oligonucleotidepair was then ligated into the NcoI and EcoRV sites of pPX1604. Theligation mixture was used to transform E. coli strain JM105.Transformants were screened for expression of M24 and LT-B by dot blotanalysis using rabbit antisera against a synthetic peptide of M24, SM24(1-29)C, and rabbit antiserum against purified LT-B. The purified LT-Bwas kindly provided by Dr. John Clements, Tulane University.

[0105] For high level expression of the fusion protein, the LT-B-M24gene was inserted into pK K223-3 (Pharmacia, Uppsala, Sweden). Thehybrid gene was cut from pPX1604 with EcoRI and SafI and the fragmentwas purified by cutting from agarose gels. Klenow fragment was used toend repair the purified DNA which was then cut with EcoRI and ligatedinto the EcoRI and SmaI restriction enzyme sites of pK K223-3. Theligation mixture was used to transform JM105 and expression of theLT-B-M24 hybrid protein was detected by colony blots as described above.One positive transformant harboring pKK223-3 that contained the hybridLT-B-M24 gene (pEC.LT-B-M24) was selected for expression andpurification of the recombinant protein.

[0106] DNA Sequencing.

[0107] The LT-B-M24 gene (SEQ ID NO:1) was sequenced usingdouble-stranded plasmid DNA and ³²P-labeled dNTPs by the dideoxychain-termination method of Sanger. Synthetic oligonucleotides copyingthe sense strand of pK K223-3 at the EcoR1 site, bases 289-303 of theLT-B gene, and the antisense strands of the HindIII site of pK K223-3provided sequence data that confirmed the location of the start codon ofthe LT-B component of the gene, the position of the M24 syntheticoligonucleotide pairs and the NcoI site.

RESULTS

[0108] Immunogenicity of LT-B-M24 Hybrid Protein.

[0109] Rabbits immunized with LT-B-M24 developed high titers ofantibodies against LT-B, pep M24 and SM24 (1-12)C, as determined byELISA (Table 1). Interestingly, the antibody titers against thesynthetic peptide were equivalent to those against the native pep M24,suggesting that the majority of the M24 antibodies evoked by theLT-B-M24 hybrid recognized M protein epitopes in the native molecule.Immune sera from all three rabbits opsonized type 24 streptococci,indicating that the M protein antibodies were directed againstprotective epitopes of the surface M protein (Table 1). None of theantisera cross-reacted with human myocardial tissue (data not shown).ELISAs performed at various intervals after the initial injection ofLT-B-M24 revealed a brisk antibody response in all three rabbits, evenafter a single intracutaneous does of LT-B-M24 (FIG. 3).

[0110] The rabbit antisera raised against LT-B-M24 containedtype-specific, bactericidal antibodies against type 24 streptococci(Table 2). All three antisera had significant bactericidal activityagainst type 24 streptococci, which in some instances was equivalent tothat observed with antiserum against intact pep M24. None of theantisera had bactericidal activity against type 5 streptococci,indicating the type-specificity of the M24 epitopes included in theLT-B-M24 hybrid protein. Passive mouse protection tests performed withantisera from rabbit #9146 indicated that antibodies against LT-B-M24provided significant protection from death compared to pre-immune serumafter intraperitoneal challenge with type 24 streptococci (Table 3). Ina separate experiment, the LD₅₀ of type 24 streptococci in this assaywas 1.5×10⁵ CFU after intraperitoneal injections of preimmune serum,whereas the LD₅₀ after giving LT-B-M24 antiserum was 2.5×10⁶.

[0111] It is to be understood that the examples and embodimentsdescribed above are not limiting and are for illustrative purposes onlyand that various modifications or changes in light thereof will besuggested to persons skilled in the art and are to be included withinthe spirit and purview of this application and the scope of he appendedclaims.

1 19 1 417 DNA Artificial Sequence LT-B-M24 hybrid molecule 1 atgaataaagtaaaatgtta tgttttattt acggcgttac tatcctctct atgtgcatac 60 ggagctccccagtctattac agaactatgt tcggaatatc gcaacacaca aatatatacg 120 ataaatgacaagatactatc atatacggaa tcgatggcag gcaaaagaga aatggttatc 180 attacatttaagagcggcgc aacatttcag gtcgaagtcc cgggcagtca acatatagac 240 tcccaaaaaaaagccattga aaggatgaag gacacattaa gaatcacata tctgaccgag 300 accaaaattgataaattatg tgtatggaat aataaaaccc ccaattcaat tgcggcaatc 360 agtatggaaaaccatggagt cgcgactagg tctcagacag atactctgga aaaataa 417 2 138 PRTArtificial Sequence LT-B-M24 hybrid molecule 2 Met Asn Lys Val Lys CysTyr Val Leu Phe Thr Ala Leu Leu Ser Ser 1 5 10 15 Leu Cys Ala Tyr GlyAla Pro Gln Ser Ile Thr Glu Leu Cys Ser Glu 20 25 30 Tyr Arg Asn Thr GlnIle Tyr Thr Ile Asn Asp Lys Ile Leu Ser Tyr 35 40 45 Thr Glu Ser Met AlaGly Lys Arg Glu Met Val Ile Ile Thr Phe Lys 50 55 60 Ser Gly Ala Thr PheGln Val Glu Val Pro Gly Ser Gln His Ile Asp 65 70 75 80 Ser Gln Lys LysAla Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Thr 85 90 95 Tyr Leu Thr GluThr Lys Ile Asp Lys Leu Cys Val Trp Asn Asn Lys 100 105 110 Thr Pro AsnSer Ile Ala Ala Ile Ser Met Glu Asn His Gly Val Ala 115 120 125 Thr ArgSer Gln Thr Asp Thr Leu Glu Lys 130 135 3 765 DNA Artificial Sequence Anantigen of M5 and a carrier of the COOH-terminal portion of M5 3atggtcgcga ctaggtctca gacagatact ctggaaaaag tacaagaagg atccaacaaa 60atttcagacg caagccgtaa gggtcttcgt cgtgacttag acgcatcgcg tgaagctaag 120aagcaattag aagctgaaca ccaaaaactt gaagaacaaa acaagatttc agaagcaagt 180cgcaaaggcc ttcgccgtga tttagacgca tcacgtgaag ctaagaagca attagaagct 240gaacaacaaa aacttgaaga acaaaacaag atttcagaag caagtcgcaa aggccttcgc 300cgtgatttag acgcatcacg tgaagctaag aaacaagttg aaaaagcttt agaagaagca 360aacagcaaat tagctgctct tgaaaaactt aacaaagagc ttgaagaaag caagaaatta 420acagaaaaag aaaaagctga gctacaagca aaacttgaag cagaagcaaa agcactcaaa 480gaacaattag caaaacaagc tgaagaactt gcaaaactaa gagctggaaa agcatcagac 540tcacaaaccc ctgatacaaa accaggaaac aaagctgttc caggtaaagg tcaagcacca 600caagcaggta caaaaccaaa ccaaaacaaa gcaccaatga aggaaactaa gagacagtta 660ccatcaacag gtgaaacagc taacccattc ttcacagcgg cagcccttac tgttatggca 720acagctggag tagcagcagt tgtaaaacgc aaagaagaaa attaa 765 4 254 PRTArtificial Sequence An antigen of M5 and a carrier of the COOH-terminalportion of M5 4 Met Val Ala Thr Arg Ser Gln Thr Asp Thr Leu Glu Lys ValGln Glu 1 5 10 15 Gly Ser Asn Lys Ile Ser Asp Ala Ser Arg Lys Gly LeuArg Arg Asp 20 25 30 Leu Asp Ala Ser Arg Glu Ala Lys Lys Gln Leu Glu AlaGlu His Gln 35 40 45 Lys Leu Glu Glu Gln Asn Lys Ile Ser Glu Ala Ser ArgLys Gly Leu 50 55 60 Arg Arg Asp Leu Asp Ala Ser Arg Glu Ala Lys Lys GlnLeu Glu Ala 65 70 75 80 Glu Gln Gln Lys Leu Glu Glu Gln Asn Lys Ile SerGlu Ala Ser Arg 85 90 95 Lys Gly Leu Arg Arg Asp Leu Asp Ala Ser Arg GluAla Lys Lys Gln 100 105 110 Val Glu Lys Ala Leu Glu Glu Ala Asn Ser LysLeu Ala Ala Leu Glu 115 120 125 Lys Leu Asn Lys Glu Leu Glu Glu Ser LysLys Leu Thr Glu Lys Glu 130 135 140 Lys Ala Glu Leu Gln Ala Lys Leu GluAla Glu Ala Lys Ala Leu Lys 145 150 155 160 Glu Gln Leu Ala Lys Gln AlaGlu Glu Leu Ala Lys Leu Arg Ala Gly 165 170 175 Lys Ala Ser Asp Ser GlnThr Pro Asp Thr Lys Pro Gly Asn Lys Ala 180 185 190 Val Pro Gly Lys GlyGln Ala Pro Gln Ala Gly Thr Lys Pro Asn Gln 195 200 205 Asn Lys Ala ProMet Lys Glu Thr Lys Arg Gln Leu Pro Ser Thr Gly 210 215 220 Glu Thr AlaAsn Pro Phe Phe Thr Ala Ala Ala Leu Thr Val Met Ala 225 230 235 240 ThrAla Gly Val Ala Ala Val Val Lys Arg Lys Glu Glu Asn 245 250 5 855 DNAArtificial Sequence An antigen of three segments of M5 and a carrier ofthe COOH-terminal portion of M5 5 atggtcgcga ctaggtctca gacagatactctggaaaaag tacaagaagt cgcgactagg 60 tctcagacag atactctgga aaaagtacaagaagtcgcga ctaggtctca gacagatact 120 ctggaaaaag tacaagaagg attcaacaaaatttcagacg caagccgtaa gggtcttcgt 180 cgtgacttag acgcatcgcg tgaagctaagaagcaattag aagctgaaca ccaaaaacct 240 gaagaacaaa acaagatttc agaagcaagtcgcaaaggcc ttcgccgtga tttagacgca 300 tcacgtgaag ctaagaagca attagaagctgaacaacaaa aacttgaaga acaaaacaag 360 atttcagaag caagtcgcaa aggccttcgccgtgatttag acgcatcacg tgaagctaag 420 aaacaagttg aaaaagcttt agaagaagcaaacagcaaat tagctgctct tgaaaaactt 480 aacaaagagc ttgaagaaag caagaaattaacagaaaaag aaaaagctga gctacaagca 540 aaacttgaag cagaagcaaa agcactcaaagaacaattag caaaacaagc tgaagaactt 600 gcaaaactaa gagctggaaa agcatcagactcacaaaccc ctgatacaaa accaggaaac 660 aaagctgttc caggtaaagc tcaagcaccacaagcaggta caaaaccaaa ccaaaacaaa 720 gcaccaatga aggaaactaa gagacagttaccatcaacag gtgaaacagc taacccattc 780 ttcacagcgg cagcccttac tgttatggcaacagctggag tagcagcagt tgtaaaacgc 840 aaagaagaaa attaa 855 6 284 PRTArtificial Sequence An antigen of three fragments of M5 and a carrier ofthe COOH-terminal portion of M5 6 Met Val Ala Thr Arg Ser Gln Thr AspThr Leu Glu Lys Val Gln Glu 1 5 10 15 Val Ala Thr Arg Ser Gln Thr AspThr Leu Glu Lys Val Gln Glu Val 20 25 30 Ala Thr Arg Ser Gln Thr Asp ThrLeu Glu Lys Val Gln Glu Gly Phe 35 40 45 Asn Lys Ile Ser Asp Ala Ser ArgLys Gly Leu Arg Arg Asp Leu Asp 50 55 60 Ala Ser Arg Glu Ala Lys Lys GlnLeu Glu Ala Glu His Gln Lys Pro 65 70 75 80 Glu Glu Gln Asn Lys Ile SerGlu Ala Ser Arg Lys Gly Leu Arg Arg 85 90 95 Asp Leu Asp Ala Ser Arg GluAla Lys Lys Gln Leu Glu Ala Glu Gln 100 105 110 Gln Lys Leu Glu Glu GlnAsn Lys Ile Ser Glu Ala Ser Arg Lys Gly 115 120 125 Leu Arg Arg Asp LeuAsp Ala Ser Arg Glu Ala Lys Lys Gln Val Glu 130 135 140 Lys Ala Leu GluGlu Ala Asn Ser Lys Leu Ala Ala Leu Glu Lys Leu 145 150 155 160 Asn LysGlu Leu Glu Glu Ser Lys Lys Leu Thr Glu Lys Glu Lys Ala 165 170 175 GluLeu Gln Ala Lys Leu Glu Ala Glu Ala Lys Ala Leu Lys Glu Gln 180 185 190Leu Ala Lys Gln Ala Glu Glu Leu Ala Lys Leu Arg Ala Gly Lys Ala 195 200205 Ser Asp Ser Gln Thr Pro Asp Thr Lys Pro Gly Asn Lys Ala Val Pro 210215 220 Gly Lys Ala Gln Ala Pro Gln Ala Gly Thr Lys Pro Asn Gln Asn Lys225 230 235 240 Ala Pro Met Lys Glu Thr Lys Arg Gln Leu Pro Ser Thr GlyGlu Thr 245 250 255 Ala Asn Pro Phe Phe Thr Ala Ala Ala Leu Thr Val MetAla Thr Ala 260 265 270 Gly Val Ala Ala Val Val Lys Arg Lys Glu Glu Asn275 280 7 15 PRT Artificial Sequence NH2-terminal fragment of M proteinfor constructing antigens, which elicit opsonic antibodies in animmunized animal 7 Val Ala Thr Arg Ser Gln Thr Asp Thr Leu Glu Lys ValGln Glu 1 5 10 15 8 15 PRT Artificial Sequence NH2-terminal fragment ofM protein for constructing antigens, which elicit opsonic antibodies inan immunized animal 8 Ala Val Thr Arg Gly Thr Ile Asn Asp Pro Gln ArgAla Lys Glu 1 5 10 15 9 15 PRT Artificial Sequence NH2-terminal fragmentof M protein for constructing antigens, which elicit opsonic antibodiesin an immunized animal 9 Arg Val Phe Pro Arg Gly Thr Val Glu Asn Pro AspLys Ala Arg 1 5 10 15 10 15 PRT Artificial Sequence NH2-terminalfragment of M protein for constructing antigens, which elicit opsonicantibodies in an immunized animal 10 Arg Val Arg Tyr Thr Arg His Thr ProGlu Asp Lys Leu Lys Lys 1 5 10 15 11 15 PRT Artificial SequenceNH2-terminal fragment of M protein for constructing antigens, whichelicit opsonic antibodies in an immunized animal 11 Asp Ala Arg Ser ValAsn Gly Glu Phe Pro Arg His Val Lys Leu 1 5 10 15 12 15 PRT ArtificialSequence NH2-terminal fragment of M protein for constructing antigens,which elicit opsonic antibodies in an immunized animal 12 Asn Gly AspGly Asn Pro Arg Glu Val Ile Glu Asp Leu Ala Ala 1 5 10 15 13 15 PRTArtificial Sequence NH2-terminal fragment of M protein for constructingantigens, which elicit opsonic antibodies in an immunized animal 13 AlaPro Leu Thr Arg Ala Thr Ala Asp Asn Lys Asp Glu Leu Ile 1 5 10 15 14 15PRT Artificial Sequence NH2-terminal fragment of M protein forconstructing antigens, which elicit opsonic antibodies in an immunizedanimal 14 His Ser Asp Leu Val Ala Glu Lys Glu Arg Leu Glu Asp Leu Gly 15 10 15 15 7 PRT Artificial Sequence Proline rich linker for the desiredM protein that links the carrier and fragment in tandem 15 Pro Gly AsnPro Ala Val Pro 1 5 16 7 PRT Artificial Sequence Proline rich linker forthe desired M protein that links the carrier and fragment in tandem 16Asp Pro Arg Val Pro Ser Ser 1 5 17 6 PRT Artificial Sequence Hexapeptideconsensus sequence 17 Leu Pro Ser Thr Gly Glu 1 5 18 9 PRT ArtificialSequence Proline rich linker for the desired M protein that links thecarrier and fragment in tandem 18 Pro Gly Pro Gly Gly Ala Pro Leu Gly 15 19 4 PRT Artificial Sequence Tetrapeptide 19 Asn Lys Ile Ser 1

I claim:
 1. A recombinant hybrid Streptococcal M protein antigen,comprising a carrier fused to at least one amino-terminal peptidefragment of streptococcal M protein having an epitope that elicitsopsonic antibodies to at least one group A streptococci serotype withouteliciting cross-reactive antibodies to mammalian tissue antigens.
 2. Theantigen of claim 1 wherein the amino-terminal peptide_fragment ofstreptococcal M protein contains 10 amino acids to 35 amino acids. 3.The antigen of claim 2 wherein the amino-terminal peptide fragment ofstreptococcal M protein contains 15 amino acids.
 4. The antigen of claim1 wherein the carrier elicits mucosal antibodies.
 5. The antigen ofclaim 1, wherein the carrier is free of an epitope that elicitsantibodies to a serotype of streptococcal M protein.
 6. The antigen ofclaim 5 wherein the carrier is a B subunit of E. coli labile toxin. 7.The antigen of claim 5 wherein the carrier has a C-repeat portion of aStreptococcal M protein.
 8. The antigen of claim 5, wherein the carrieris a carboxy-terminal portion of a Streptococcal M protein.
 9. Theantigen of claim 8, wherein the carboxy-terminal portion is of M5. 10.The antigen of claim 5, wherein the carrier is the carboxy-terminalportion of a surface protein from a Gram-positive cocci.
 11. The antigenof claim 5, wherein the carrier is selected from the group consisting oftetanus toxoid, diphtheria toxoid, bovine serum albumin, hen egglysozyme, gelatin, bovine gamma globulin, B subunit of cholera toxin, Bsubunit of E. coli labile toxin and flagellin polymer.
 12. The antigenof claim 1 wherein the carrier and said at least one amino-terminalpeptide fragment of streptococcal M protein are linked in tandem by alinker comprising amino acids.
 13. The antigen of claim 12, wherein theamino acids of the linker are encoded by a nucleotide sequencecomprising a restriction enzyme site.
 14. The antigen of claim 12wherein the linker ranges in size from 1 amino acid to 30 amino acids.15. The antigen of claim 12, wherein the linker ranges in size from 2amino acids to 7 amino acids.
 16. The antigen of claim 12, wherein thelinker comprises hydrophobic amino acids.
 17. The antigen of claim 16,wherein the hydrophobic amino acids are selected from the groupconsisting of tryptophan, alanine, leucine, isoleucine, valine,tyrosine, phenylalanine, proline, methionine and combinations thereof.18. The antigen of claim 12, wherein the linker is proline-rich.
 19. Theantigen of claim 16, wherein the linker is selected from the groupconsisting of Ile-Pro-Gly, Pro-Gly-Asn-Pro-Ala-Val-Pro (SEQ ID NO: 15),and Asp-Pro-Arg-Val-Pro-Ser-Ser (SEQ ID NO: 16).
 20. The antigen ofclaim 18, wherein the linker has two or three prolines and two or threeglycines.
 21. The antigen of claim 12, wherein the linker is His-Gly orGly-Ser.
 22. The antigen of claim 1 wherein said serotype of Group Astreptococcal M protein is at least one of serotype M 1, M3, M12, M18,or M
 19. 23. The antigen of claim 12 wherein said serotype of Group Astreptococcal M protein is at least one of serotype M1, M3, M12, M18, orM19.
 24. The antigen of claim 1 wherein at least one serotype is fromrheumatogenic streptococci.
 25. The antigen of claim 12 wherein at leastone serotype is from rheumatogenic streptococci.
 26. A composition,comprising a biologically acceptable diluent or adjuvant and the antigenaccording to any one of claims 1-25.
 27. A cocktail composition,comprising at least two recombinant hybrid Streptococcal M proteinantigens according to any one of claims 1-25.
 28. A nucleic acidmolecule comprising a nucleotide sequence encoding a recombinant hybridStreptococcal M protein antigen according to any one of claims 1-25. 29.A nucleic acid expression construct comprising the nucleic acid moleculeaccording to claim 28, wherein a host cell containing the nucleic acidexpression construct can express the recombinant hybrid Streptococcal Mprotein antigen encoded by the nucleic acid molecule.
 30. A host cellcomprising a nucleic acid expression construct according to claim 29.31. A method of producing a recombinant hybrid Streptococcal M proteinantigen, comprising growing a host cell according to claim 30 underconditions that allow expression of a recombinant hybrid Streptococcal Mprotein antigen, and isolating the expressed recombinant hybridStreptococcal M protein antigen.
 32. A recombinant hybrid StreptococcalM protein antigen produced by the method of claim
 31. 33. A method foreliciting opsonic antibodies to at least one group A streptococciwithout eliciting cross-reactive antibodies to mammalian tissueantigens, comprising administering to a patient in need thereof aneffective amount of a composition comprising a biologically acceptablediluent and a non-virulent, live host cell containing a nucleic acidexpression construct comprising the nucleic acid molecule according toclaim 28, wherein a host cell containing the nucleic acid expressionconstruct can express the recombinant hybrid Streptococcal M proteinantigen encoded by the nucleic acid molecule.
 34. The method of claim 33wherein the expressed recombinant hybrid Streptococcal M protein antigenis compartmentalized intracellularly, wherein release of the antigenfrom a host cell elicits opsonic antibodies.
 35. The method of claim 33wherein the carrier is the carboxy-terminal portion of a surface of agram-positive cocci.
 36. The method of claim 33 wherein the host cell isa bacterium, the bacterium being a non-cariogenic Streptococcus mutans.37. The method of claim 33 wherein the composition is administeredorally, intranasally, or mucosally.
 38. A method for eliciting opsonicantibodies to at least one group A streptococci serotype withouteliciting cross-reactive antibodies to mammalian tissue antigens,comprising administering to a patient in need thereof an effectiveamount of the composition according to claim
 26. 39. A method foreliciting opsonic antibodies to at least one group A streptococciserotype without eliciting cross-reactive antibodies to mammalian tissueantigens, comprising administering to a patient in need thereof aneffective amount of the cocktail composition according to claim
 27. 40.The method for eliciting opsonic antibodies according to claim 35wherein the composition is administered orally, intranasally, ormucosally.
 41. The method for eliciting opsonic antibodies according toclaim 39 wherein the composition is administered orally, intranasally,or mucosally.